Methods and compositions for treatment of angiogenic disorders using anti-VEGF agents

ABSTRACT

Provided are methods and compositions for treatment of angiogenic disorders using anti-VEGF agents. The anti-VEGF agents comprise VEGF binding domains and have the ability to bind vitreous. Provided are exemplary embodiments of Fc-IgG fusion proteins with VEGF binding domains with strong heparin-binding characteristics, strong inhibition of VEGF mitogenic activity, and improved pharmacokinetics, namely longer half-lives of the anti-VEGF agents and consequently less frequent dosing.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of PCT/US2019/015160 filedon Jan. 25, 2019 which claims the priority benefit of U.S. ProvisionalApplication No. 62/622,382, filed Jan. 26, 2018, which applications areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 25, 2019, isnamed 24978-0470_SL.txt and is 50,960 bytes in size.

BACKGROUND

The development of a neovascular supply or angiogenesis serves crucialhomeostatic roles since the blood vessels carry nutrients to tissues andorgans and remove catabolic products¹. However, uncontrolled growth ofblood vessels can promote or facilitate numerous disease processes,including tumors and intraocular vascular disorders¹. Although severalangiogenic factors were initially identified and characterized (e.g.,EGF, TGF-α, TGF-β, aFGF, bFGF, angiogenin)², work conducted over thelast three decades has established the critical role of VEGF-A (VEGFhereafter) in normal and pathological angiogenesis³ ⁴. VEGF is a memberof a gene family that also includes PlGF, VEGF-B, VEGF-C and VEGF-D.Three related receptor tyrosine kinase (RTKs) have been reported to bindVEGF ligands: VEGFR-1, VEGFR-2 and VEGFR-3⁵. P1GF and VEGF-B interactselectively with VEGFR-1, VEGF binds both VEGFR-1 and VEGFR-2. A thirdmember of this family of RTKs, VEGFR-3⁶, binds VEGF-C and VEGF-D, whichare implicated in lymphangiogenesis. Each member of this RTK class hasseven immunoglobulin (Ig)-like domains in the extracellular portion⁷.There is agreement that VEGFR-2 is the main signaling receptor forVEGF⁵. However, VEGFR-1 binds VEGF with substantially higher bindingaffinity than VEGFR-2⁷.

VEGF inhibitors have become a standard of therapy in multiple tumors andhave revolutionized the treatment of intraocular neovascular disorderssuch as the neovascular form of age-related macular degeneration (AMD),proliferative diabetic retinopathy and retinal vein occlusion, which areleading causes of severe vision loss and legal blindness⁸ ⁴. Currently,three anti-VEGF drugs are widely used in the USA for ophthalmologicalindications: bevacizumab, ranibizumab and aflibercept⁴. Bevacizumab is afull-length IgG antibody targeting VEGF⁹. Even though bevacizumab wasnot developed for ophthalmological indications, it is widely usedoff-label due to its low cost. Ranibizumab is an affinity-maturedanti-VEGF Fab¹⁰. Aflibercept is an IgG-Fc fusion protein¹¹ ¹², withelements from VEGFR-1 and VEGFR-2, that binds VEGF, PIGF and VEGF-B¹³Conbercept is a soluble VEGF receptor structurally related toaflibercept, widely used as treatment of intraocular neovascularizationin China¹⁴. Millions of patients worldwide have been treated with thesedrugs. Importantly, after five-year treatment with ranibizumab orbevacizumab, about half of neovascular AMD patients had good vision,i.e. visual acuity 20/40 or better, an outcome that would have been outof reach before anti-VEGF agents were available¹⁵.

However, in real-life clinical settings, many patients receive feweranti-VEGF injections than in clinical trials and it has beenhypothesized that this may correlate with poor visual outcomes¹⁶.Indeed, the need to perform relatively frequent intravitreal injectionshas hampered patient compliance and ultimately the benefits of thetherapy, especially in some countries¹⁶. Therefore, there is a need todevelop agents with longer duration when injected in the eye, thusreducing the frequency of injections and a number of approaches to thisend have been attempted^(17,18). Aflibercept (EYLEA) was approved basedon clinical trials showing that every 8-week administration of the doseof 2 mg could match the efficacy of monthly ranibizumab (0.5 mg).However, despite the prediction that a switch to aflibercept wouldreduce the number of intravitreal injections, recent studies suggestthat it is not the case¹⁹. Therefore, there is still an unmet medicalneed for intravitreal anti-VEGF agents with improved half-life.

In 1996 Davis-Smyth et al²⁰ (see also U.S. Pat. No. 5,952,199) reportedthat domain (D) 2 of VEGFR-1 is the critical binding element for VEGFand PIGF. Deletion of D2 completely abolished binding. Replacing D2 ofVEGFR-3 with VEGFR-1 D2 conferred on VEGFR-3 the ligand specificity ofVEGFR-1²⁰. Subsequent work documented the interaction between D2 andVEGF (or PlGF) by X-ray crystallography²¹⁻²³.

The initial studies led to the design of a construct with full VEGFbinding characteristics, comprising the first three Ig-like Ds ofVEGFR-1, fused to an Fc-IgG (Flt-1-3-IgG)²⁰. Flt-1-3-IgG showed a potentability to neutralize VEGF, in vitro and in vivo²⁴⁻²⁷ However, thesystemic half-life of this molecule was hampered by the presence of D3,which has significant heparin affinity due to the presence of clustersof basic residues, resulting in binding to HSPGs in various tissues. In2002 Holash et al¹³ (U.S. Pat. No. 7,070,959) described an IgG fusionconstruct comprising of VEGFR-1 D2 (the binding element) and D3 ofVEGFR2, which has much weaker heparin affinity compared to VEGFR-1 D3.This molecule, known today as aflibercept (marketed as EYLEA), wasreported to have a longer half-life compared to Flt-(1-3-IgG) followingsystemic administration¹³, clearly an advantage for treatment aiming,for example, at oncological indications.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for inhibitingangiogenesis and for treating VEGF-associated conditions, such as oculardisease, including but not limited to, age-related macular degeneration,proliferative diabetic retinopathy, retinal vein occlusion, choroidalneovascularization secondary to myopia, retinopathy of prematurity,diabetic macular edema, polypoidal choroidal vasculopathy, comprisingadministering an anti-VEGF agent that inhibits the activity of VEGF and,at the same time, has strong heparin-binding characteristics, therebyproviding superior pharmacokinetics, namely having a longer half-life ofthe therapeutic agent following intravitreal administration.

In embodiments, the present invention provides compositions and methodsof treating conditions in which local direct administration of ananti-VEGF agent is beneficial, for example, treating and preventingendothelial cell proliferative conditions or angiogenesis, for example,in treating solid tumors, such as but not limited to, intracranialadministration in glioblastomas.

In embodiments, the present invention provides an anti-VEGF agent,wherein the anti-VEGF agent is an Fc-IgG construct fusing domains withVEGF binding characteristics and domains that bind heparinproteoglycans. In embodiments, the present invention provides ananti-VEGF agent, wherein the anti-VEGF agent is an Fc-IgG constructhaving the ability to bind heparin and contains one or more domains withVEGF binding characteristics. In embodiments, the present inventionprovides an anti-VEGF agent, wherein the anti-VEGF agent is a fusionprotein with improved efficacy for binding to VEGF and heparin. Inembodiments, the present invention provides an anti-VEGF agent, whereinthe anti-VEGF agent is a fusion protein with very low endotoxin levels.

In embodiments, the present invention provides an anti-VEGF agent,wherein the anti-VEGF agent is an IgG chimeric protein comprisingelements of VEGF receptors. In embodiments, the present inventionprovides an IgG chimeric protein, wherein the IgG chimeric proteincomprises one or more fragments of the seven immunoglobulin (Ig)-likedomains in the extracellular portion of VEGF tyrosine kinase receptors.In embodiments, the present invention provides an IgG chimeric protein,wherein the IgG chimeric protein comprises one or more extracellulardomain fragments of VEGFR-1 fused with Fc-IgG. In embodiments, thepresent invention provides an IgG chimeric protein comprising at leastone VEGF binding domain VEGFR-1 domain 2 and at least one additionalVEGFR-1 domain 1 or 3, and not including domain 4. In embodiments, thepresent invention provides an IgG chimeric protein, wherein the IgGchimeric protein comprises one or more extracellular domain fragments ofVEGFR-2 fused with Fc-IgG. In embodiments, the present inventionprovides an IgG chimeric protein, wherein the IgG chimeric proteincomprises one or more extracellular domain fragments of VEGFR-1 andVEGFR-2 fused with Fc-IgG.

In embodiments, the present invention provides an anti-VEGF agentcomprising a VEGF binding portion operatively linked to a Fc-IgG,wherein the VEGF binding portion comprises at least one VEGF bindingdomain that is an IgG-like domain 2 of VEGFR-1, and wherein theanti-VEGF agent has a VEGF-stimulated mitogenesis-inhibiting abilitygreater than aflibercept. In embodiments, the present invention providesthat the anti-VEGF agent has a vitreous binding ability greater thanaflibercept. In embodiments, the present invention provides that theanti-VEGF agent has a vitreous bound VEGF-stimulated endothelial cellproliferation-inhibiting ability greater than aflibercept. Inembodiments, the present invention provides that the agent has anincreased half-life in vivo compared to aflibercept.

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 1, 2, and 3 of VEGFR-1(V₁₋₂₋₃). In embodiments, the anti-VEGF agent comprises amino acidsequence as defined in SEQ ID NO: 1.

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 2 and 3 of VEGFR-1(V₂₋₃). In embodiments, the anti-VEGF agent comprises amino acidsequence as defined in SEQ ID NO: 3.

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 1, 2, 3 and 3 ofVEGFR-1 (V₁₋₂₋₃₋₃). In embodiments, the anti-VEGF agent comprises aminoacid sequence as defined in SEQ ID NO: 5.

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 2, 3 and 3 of VEGFR-1(V₂₋₁₋₃). In embodiments, the the anti-VEGF agent comprises amino acidsequence as defined in SEQ ID NO: 7.

In embodiments, the present invention provides pharmaceuticalcompositions comprising a therapeutically effective amount of ananti-VEGF agent as defined claims and a pharmaceutically acceptableexcipient. In embodiments, the present invention provides methods oftreating a VEGF-related disorder in a subject in need comprisingadministering to the subject a therapeutically effective amount of ananti-VEGF agent as defined. The anti-VEGF agent can be directly injectedinto the affected tissue or organ, such as an eye.

In embodiments, the present invention provides a method for treatingocular disease, wherein an anti-VEGF agent is administered locally tothe eye at a dosage corresponding to a molar ratio of 2:1 compared toVEGF. In embodiments, the present invention provides a method fortreating ocular disease, wherein an anti-VEGF agent is administered byintravitreous injection. In embodiments, the present invention providesa method for treating ocular disease, wherein an anti-VEGF agent isadministered every 4-6 weeks, and in other embodiments, the treatment iscontinued for a period of at least one year.

According to one embodiment, the present invention provides a method fortreating ocular disease comprising administering a therapeuticallyeffective amount of an anti-VEGF agent locally into the eye wherein thetreatment is effective to treat occult, minimally classic, andpredominantly classic forms of wet macular degeneration, wherein theagent is a fusion protein.

In embodiments the invention can be used to treat a wide variety ofVEGF-related disorders including neovascular age related maculardegeneration, choroidal neovascularization secondary to myopia,proliferative diabetic retinopathy, diabetic macular edema, retinalvascular obstruction such as retinal vein occlusion, ocular tumors, vonHippel Lindau syndrome, retinopathy of prematurity, polypoid choroidalvasculopathy, colorectal cancer, lung cancer, cervical cancer,endometrial cancer, ovarian cancer, kidney cancer, schwannomas, gliomas,ependimomas, and neoplastic or non-neoplastic disorders that benefitfrom anti-VEGF therapy.

According to another aspect, the present invention provides apharmaceutical formulation comprising an anti-VEGF agent in apharmaceutically acceptable carrier formulation for local administrationsuch as into the eye.

In embodiments, the present invention discloses novel constructs,wherein the constructs potently neutralize the activity of VEGF while,at the same time, have strong heparin-binding characteristics.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the presentdisclosure will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the disclosure are utilized, and the accompanying drawingsof which:

FIG. 1 depicts a schematic representation of exemplary constructedfusion proteins with various Ig-like extracellular domains of VEGFR-1(V) fused to Fc-IgG (Fc). The following constructs are shown: V₁₋₂₋₃-Fc;V₂₋₃-Fc; V₁₋₂₋₃₋₃-Fc; V₂₋₃₋₃-Fc; V₁₋₂₋₃₋₄-Fc; V₂₋₃₋₄-Fc; V₁₋₂₋₄-Fc andV₂₋₄-Fc.

FIG. 2 depicts a strategy of plasmid construction and expression.

FIG. 3 depicts the amino acid sequence and nucleic acid sequence ofconstruct V₁₋₂₋₃. SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

FIG. 4 depicts the amino acid sequence and nucleic acid sequence ofconstruct V₂₋₃. SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

FIG. 5 depicts the amino acid sequence and nucleic acid sequence ofconstruct V₁₋₂₋₃₋₃. SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

FIG. 6 depicts the amino acid sequence and nucleic acid sequence ofconstruct V₂₋₃. SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

FIG. 7 depicts the amino acid sequence and nucleic acid sequence ofconstruct V₁₋₂₋₃₋₃₋₄. SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

FIG. 8 depicts the amino acid sequence and nucleic acid sequence ofconstruct V₂₋₃₋₄. SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

FIG. 9 depicts the amino acid sequence and nucleic acid sequence ofconstruct V₂₋₄. SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

FIG. 10 depicts the expression of VEGFR-1 constructs in 293 cells.

FIG. 11 depicts silver-stained PAGE gels under reducing and non-reducingconditions of 200 ng of each VEGFR-1 Fc fusion protein compared toEYLEA.

FIG. 12 depicts inhibitory effects of VEGF receptor chimeric proteins onVEGF-stimulated endothelial cell proliferation.

FIG. 13 depicts competition of VEGF for Biotinylated VEGF (at 100 ng/ml)binding to VEGFR1 soluble receptor.

FIG. 14 depicts VEGFR-1 soluble receptor binding to Biotinylated VEGFand bovine vitreous.

FIG. 15 depicts bovine vitreous-bound V₁₋₂₋₃₋₃ is biologically active.

FIG. 16 shows effects of control IgG, EYLEA, or VEGFR-1 Fc fusionproteins on choroidal neovascularization (CNV) area. Each protein wasinjected intravitreally in the mouse at the dose of 2.5 mg one daybefore laser treatment. EYLEA was tested also at 25 mg. Asterisks denotesignificant differences (Student's t test) compared to the appropriateIgG control groups (**p<0.01, *p<0.05).

FIGS. 17A and 17B show effects of EYLEA, V_(1,2,3,3) or control IgG onCNV area following a single intravitreal administration (2.5 mg), 1 day,7 days or 14 days before laser treatment. Asterisk denote significantdifferences (p<0.05, Student's t test) compared to the IgG controlgroup. FIG. 17B shows representative CD31 immunofluorescence images ofgroups in FIG. 17A.

DETAILED DESCRIPTION

All publications, patents, and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

It is understood that aspects and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments. Other objects, advantages and features of the presentinvention will become apparent from the following specification taken inconjunction with the accompanying figures.

When introducing elements of the present invention or the preferredembodiment(s) thereof, the articles “a,” “an,” “the,” and “said” areintended to mean that there are one or more of the elements. The terms“comprising,” “including,” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains”, “containing,” “characterizedby,” or any other variation thereof, are intended to encompass anon-exclusive inclusion, subject to any limitation explicitly indicatedotherwise, of the recited components. For example, a fusion protein, apharmaceutical composition, and/or a method that “comprises” a list ofelements (e.g., components, features, or steps) is not necessarilylimited to only those elements (or components or steps), but may includeother elements (or components or steps) not expressly listed or inherentto the fusion protein, pharmaceutical composition and/or method.

As used herein, the transitional phrases “consists of” and “consistingof” exclude any element, step, or component not specified. For example,“consists of” or “consisting of” used in a claim would limit the claimto the components, materials or steps specifically recited in the claimexcept for impurities ordinarily associated therewith (i.e., impuritieswithin a given component). When the phrase “consists of” or “consistingof” appears in a clause of the body of a claim, rather than immediatelyfollowing the preamble, the phrase “consists of” or “consisting of”limits only the elements (or components or steps) set forth in thatclause; other elements (or components) are not excluded from the claimas a whole.

As used herein, the transitional phrases “consists essentially of” and“consisting essentially of” are used to define a fusion protein,pharmaceutical composition, and/or method that includes materials,steps, features, components, or elements, in addition to those literallydisclosed, provided that these additional materials, steps, features,components, or elements do not materially affect the basic and novelcharacteristic(s) of the claimed invention. The term “consistingessentially of” occupies a middle ground between “comprising” and“consisting of”.

As used herein, the term “pharmaceutical composition” contemplatescompositions comprising one or more therapeutic agents or drugs asdescribed below, and one or more pharmaceutically acceptable excipients,carriers, or vehicles.

As used herein, the term “pharmaceutically acceptable excipients,carriers, or vehicles” comprises any acceptable materials, and/or anyone or more additives known in the art. As used herein, the term“excipients,” “carriers,” or “vehicle” refer to materials suitable fordrug administration through various conventional administration routesknown in the art. Excipients, carriers, and vehicles useful hereininclude any such materials known in the art, which are nontoxic and donot interact with other components of the composition in a deleteriousmanner, and generally refers to an excipient, diluent, preservative,solubilizer, emulsifier, adjuvant, and/or vehicle with which an activeagent or drug is administered. Such carriers may be sterile liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents. Antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid; andagents for the adjustment of tonicity such as sodium chloride ordextrose may also be a carrier. Methods for producing compositions incombination with carriers are known to those of skill in the art. Insome embodiments, the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art.

As used herein, the term “therapeutically effective amount” refers tothose amounts that, when administered to a particular subject in view ofthe nature and severity of that subject's condition, will have a desiredtherapeutic effect, e.g., an amount which will cure, prevent, inhibit,or at least partially arrest or partially prevent a target condition. Insome embodiments, the term “therapeutically effective amount” or“effective amount” refers to an amount of a therapeutic agent or drugthat when administered alone or in combination with an additionaltherapeutic agent or drug to a cell, tissue, organ, or subject iseffective to prevent or ameliorate ocular diseases and cancers,including, but not limited to, age-related macular degeneration,proliferative diabetic retinopathy, retinal vein occlusion, choroidalneovascularization secondary to myopia; retinopathy of prematurity,diabetic macular edema, polypoidal choroidal vasculopathy, colorectalcancer, lung cancer, breast cancer, pancreatic cancer, and prostatecancer. A therapeutically effective dose further refers to that amountof the therapeutic agent or drug sufficient to result in amelioration ofsymptoms, e.g., treatment, healing, prevention, or amelioration of therelevant medical condition, or an increase in rate of treatment,healing, prevention, or amelioration of such conditions. When applied toan individual active ingredient administered alone, a therapeuticallyeffective dose refers to that ingredient alone. When applied to acombination, a therapeutically effective dose refers to combined amountsof the active ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously.

As used herein, the terms “treating,” “treatment,” or “alleviation”refers to therapeutic treatment wherein the object is to slow down(lessen) if not cure the targeted pathologic condition or disorder orprevent recurrence of the condition. A subject is successfully “treated”if, after receiving a therapeutic amount of a therapeutic agent or drug,the subject shows observable and/or measurable reduction in or absenceof one or more signs and symptoms of the particular condition. Reductionof the signs or symptoms of a condition may also be felt by the subject.A subject is also considered treated if the subject experiences stablecondition. In some embodiments, treatment with a therapeutic agent ordrug is effective to result in the subjects being symptom-free 3 monthsafter treatment, preferably 6 months, more preferably one year, evenmore preferably 2 or more years post treatment. These parameters forassessing successful treatment and improvement in the condition arereadily measurable by routine procedures familiar to a physician ofappropriate skill in the art.

As used herein, “preventative” treatment is meant to indicate apostponement of development of a condition or a symptom of a condition,suppressing symptoms that may appear, or reducing the risk of developingor recurrence of a condition or symptom. “Curative” treatment includesreducing the severity of or suppressing the worsening of an existingsymptom, or condition.

As used herein, the term “therapeutic agent,” “anti-VEGF agent,” “fusionprotein,” “chimeric protein,” or “recombinant protein” comprises a firstpolypeptide operatively linked to a second polypeptide, wherein the“therapeutic agent,” “anti-VEGF agent,” “fusion protein,” “chimericprotein,” or “recombinant protein” inhibits the activity of VEGF.Chimeric proteins may optionally comprise a third, fourth or fifth orother polypeptide operatively linked to a first or second polypeptide.Chimeric proteins may comprise two or more different polypeptides.Chimeric proteins may comprise multiple copies of the same polypeptide.Chimeric proteins may also comprise one or more mutations in one or moreof the polypeptides. Methods for making chimeric proteins are well knownin the art. In some embodiments the term “therapeutic agent,” “fusionprotein,” “chimeric protein,” or “recombinant protein” refers to anyconstructs expressed or synthesized, including but not limited to,peptides or proteins operatively linking one or more of the Ig-likedomains or domain fragments of VEGFR-1 and/or VEGFR-2 with Fc-IgG.

The term “Ig-like domains” refers to Ig-like domains 1-7 of VEGFR-1 andVEGFR-2. The term “Ig-like domain fragments” comprise a portion of afull length domain, generally the heparin and/or VEGF binding orvariable region thereof. Examples of domain fragments include amino acidsequences comprising a segment of at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99% of the full length domain with100% sequence identity and variations thereof. Variations in the aminoacid sequences of fusion proteins are contemplated as being encompassedby the present disclosure, providing that the variations in the aminoacid sequence maintain at least 75%, more preferably at least 80%, 90%,95%, and most preferably 99%. Certain percentages in between areincluded, such as 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%sequence identity. In particular, conservative amino acid replacementsare contemplated. Conservative replacements are those that take placewithin a family of amino acids that are related in their side chains.Genetically encoded amino acids are generally divided into families: (1)acidic amino acids are aspartate, glutamate; (2) basic amino acids arelysine, arginine, histidine; (3) non-polar amino acids are alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan, and (4) uncharged polar amino acids are glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic aminoacids include arginine, asparagine, aspartate, glutamine, glutamate,histidine, lysine, serine, and threonine. The hydrophobic amino acidsinclude alanine, cysteine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan, tyrosine and valine. Other familiesof amino acids include (i) serine and threonine, which are thealiphatic-hydroxy family; (ii) asparagine and glutamine, which are theamide containing family; (iii) alanine, valine, leucine and isoleucine,which are the aliphatic family; and (iv) phenylalanine, tryptophan, andtyrosine, which are the aromatic family. For example, it is reasonableto expect that an isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid will not have a major effect on the binding or properties of theresulting molecule, especially if the replacement does not involve anamino acid within a framework site. Whether an amino acid change resultsin a functional fusion protein can readily be determined by assaying thespecific activity of the fusion protein derivative. Fragments or analogsof fusion proteins can be readily prepared by those of ordinary skill inthe art. Preferred amino- and carboxy-termini of fragments or analogsoccur near boundaries of functional domains.

As used herein, an “isolated” or “purified” fusion protein means thefusion protein is the predominant species present (i.e., on a molarbasis it is more abundant than any other individual species in thecomposition), and preferably a substantially purified fraction is acomposition wherein the fusion protein comprises at least about 50% (ona molar basis) of all macromolecular species present. Generally, apurified composition will comprise more than about 80% of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the fusion proteinis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

Values or ranges may be expressed herein as “about,” from “about” oneparticular value, and/or to “about” another particular value. When suchvalues or ranges are expressed, other embodiments disclosed include thespecific value recited, from the one particular value, and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. It will be furtherunderstood that there are a number of values disclosed therein, and thateach value is also herein disclosed as “about” that particular value inaddition to the value itself. In embodiments, “about” can be used tomean, for example, within 10% of the recited value, within 5% of therecited value, or within 2% of the recited value.

In one aspect the present invention discloses a composition comprising atherapeutic agent, where the therapeutic agent comprises one or moreheparin binding domains of VEGFR-1 or VEGFR-2, and one or more VEGFbinding domains, thereby inhibiting the binding of VEGF to its cognatereceptor.

In embodiments, the present invention provides an anti-VEGF agentcomprising a VEGF binding portion operatively linked to a Fc-IgG,wherein the VEGF binding portion comprises at least one VEGF bindingdomain that is an IgG-like domain 2 of VEGFR-1, and wherein theanti-VEGF agent has a VEGF-stimulated mitogenesis-inhibiting abilitygreater than aflibercept. In embodiments, the present invention providesthat the anti-VEGF agent has a vitreous binding ability greater thanaflibercept. In embodiments, the present invention provides that theanti-VEGF agent has a vitreous bound VEGF-stimulated endothelial cellproliferation-inhibiting ability greater than aflibercept. Inembodiments, the present invention provides that the agent has anincreased half-life in vivo compared to aflibercept.

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 1, 2, and 3 of VEGFR-1(V₁₋₂₋₃). In embodiments, the anti-VEGF agent comprises amino acidsequence as defined in SEQ ID NO: 1.

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 2 and 3 of VEGFR-1(V₂₋₃). In embodiments, the anti-VEGF agent comprises amino acidsequence as defined in SEQ ID NO: 3.

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 1, 2, 3 and 3 ofVEGFR-1 (V₁₋₂₋₃₋₃). In embodiments, the anti-VEGF agent comprises aminoacid sequence as defined in SEQ ID NO: 5.

In embodiments, the present invention provides that the VEGF bindingportion consists essentially of IgG-like domains 2, 3 and 3 of VEGFR-1(V₂₋₃₋₃). In embodiments, the anti-VEGF agent comprises amino acidsequence as defined in SEQ ID NO: 7.

In embodiments, the present invention provides pharmaceuticalcompositions comprising a therapeutically effective amount of ananti-VEGF agent as defined claims and a pharmaceutically acceptableexcipient. In embodiments, the present invention provides methods oftreating a VEGF-related disorder in a subject in need comprisingadministering to the subject a therapeutically effective amount of ananti-VEGF agent as defined. The anti-VEGF agent can be directly injectedinto the affected tissue or organ, such as an eye.

In embodiments the invention can be used to treat a wide variety ofVEGF-related disorders including neovascular age related maculardegeneration, choroidal neovascularization secondary to myopia,proliferative diabetic retinopathy, diabetic macular edema, retinalvascular obstruction such as retinal vein occlusion, ocular tumors, vonHippel Lindau syndrome, retinopathy of prematurity, polypoid choroidalvasculopathy, colorectal cancer, lung cancer, cervical cancer,endometrial cancer, ovarian cancer, kidney cancer, schwannomas, gliomas,ependimomas, and neoplastic or non-neoplastic disorders that benefitfrom anti-VEGF therapy.

In some embodiments, the therapeutic agent is in an administrable dosageform, comprising the therapeutic agent, and an additional excipient,carrier, adjuvant, solvent, or diluent.

In some embodiments, the present invention discloses a pharmaceuticalcomposition suitable for treating and/or preventatively treating asubject, wherein the therapeutic agent is contained in an amounteffective to achieve its intended purpose.

In some embodiments, the therapeutic agent or compositions disclosedherein are administered by injection. In certain embodiments, thecompositions or the therapeutic agent are injected directly into thediseased organ or tissue. In some embodiments, the therapeutic agent canbe topically administered, for example, by patch or direct applicationto the diseased organ or tissue, or by iontophoresis. The therapeuticagents may be provided in sustained release compositions, such as thosedescribed in, for example, U.S. Pat. Nos. 5,672,659 and 5,595,760. Theuse of immediate or sustained release compositions depends on the natureof the condition being treated. If the condition consists of an acute orover-acute disorder, treatment with an immediate release form will bepreferred over a prolonged release composition. Alternatively, forcertain preventative or long-term treatments, a sustained releasedcomposition may be appropriate.

The therapeutic agent may also be delivered using an implant, such asbut not limited to an intraocular implant. Such implants may bebiodegradable and/or biocompatible implants, or may be non-biodegradableimplants. The implants may be permeable or impermeable to the activeagent. The specific implants for delivery of the therapeutic agent isdependent on both the affected tissue or organ as well as the nature ofthe condition being treated. The use of such implants is well known inthe art.

The inhibitors described in this invention can be formulated innanoparticles or other drug formulations in order to provide precisedelivery to specific tissues and also provide controlled releasetherapy.

The inhibitors described in this application can be delivered not onlyas purified recombinant proteins but also by a gene therapy approach.Recombinant adeno-associated vectors (rAAVs) or other suitable vectorscan be used to deliver the VEGF inhibitor by sub-retinal or intravitrealdelivery^(43,44).

In a related aspect, the present invention provides a method fortreating a VEGF-related or neovascular disorder in a subject, whereinthe method involves administering to the subject: (a) an effectiveamount of a fusion protein capable of binding heparin and diminishing orpreventing the development of unwanted neovasculature. The fusionprotein may be combined with other anti-VEGF agents including, but arenot limited to: antibodies or antibody fragments specific to VEGF;antibodies specific to VEGF receptors; compounds that inhibit, regulate,and/or modulate tyrosine kinase signal transduction; VEGF polypeptides;oligonucleotides that inhibit VEGF expression at the nucleic acid level,for example antisense RNAs; and various organic compounds and otheragents with angiogenesis inhibiting activity.

The invention provides that heparin-binding mediated by D3 (or otherIg-like domain) of VEGFR1²⁸, while a disadvantage for systemicadministration, can confer important advantages for intravitreal (orother local) administration. Indeed, the ability to bind HGPSG, keycomponents of the extracellular matrix²⁹, promotes accumulation in thevitreous as well as retinal penetration³⁰. The invention provides aseries of VEGFR-1 Fc fusion constructs having differential abilities tointeract with HSPGs. This enables election of VEGF inhib9itors withdifferent duration/half life in the eye, which are useful underdifference clinical conditions.

The features and other details of the invention will now be moreparticularly described and pointed out in the following examplesdescribing preferred techniques and experimental results. The examplesare provided for the purpose of illustrating the invention and shouldnot be construed as limiting.

EXAMPLES

In embodiments, the present invention therefore discloses anti-VEGFagents that are novel and improve on existing anti-VEGF agents,including aflibercept, owing to high biological potency combined withstrong heparin-binding characteristics. The heparin binding ispredictive of a longer half-life and consequently reduced frequency ofadministration.

The invention provides that heparin-binding mediated by D3 (or otherIg-like domain) of VEGFR1²⁸, while a disadvantage for systemicadministration, can confer important advantages for intravitreal (orother local) administration. Indeed, the ability to bind HGPSG, keycomponents of the extracellular matrix²⁹, may promote accumulation inthe vitreous as well as retinal penetration³⁰. The invention provides aseries of VEGFR-1 Fc fusion constructs having differential abilities tointeract with HSPGs.

FIG. 1 provides a schematic representation of the constructs employedhere. FIG. 2 illustrates the vector employed and the cloning strategy.FIG. 3-9 show the nucleic acid and amino acid sequences of theconstructs generated.

The Examples show that the expression levels of most constructs werelow; V₁₋₂₋₃₋₄, V₁₋₂₋₃₋₃, V₂₋₃₋₄ and V₁₋₂₋₄ were almost completelyundetectable in the conditioned media. Previous studies had shown thatVEGF isoforms with high affinity for heparin (VEGF₁₈₉ or VEGF₂₀₆) areundetectable in the conditioned media of transfected cells, beingtightly bound to the cells surface or the extracellular matrix³¹ ³².However, they could be released in a soluble form by the addition ofheparin or heparinase, indicating that the binding site consisted ofHSPG³¹ ³² This example sought to determine whether the addition ofheparin may also affect the levels of recombinant VEGFR-1 fusionproteins. Indeed, adding heparin to the media of transfected cells in6-well plates resulted in dose-dependent increases in the concentrationsof recombinant protein in the media (FIG. 10 ).

In seeking to purify the recombinant proteins, conventional protein A(PA) affinity chromatography alone yielded a major band of the expectedmass, but with numerous minor bands, likely reflecting the interactionof the recombinant proteins with host cell-derived HSPGs and othermolecules. A protocol was developed that removed such impurities, asdescribed in Methods. A wash at high pH (for example 9.2) in thepresence of 1.2 M NaCl while the protein is bound to PA, resulted inrelease of numerous contaminants. The next step, anion exchangechromatography with Hi-Trap Q, was very effective at removing the bulkof contaminants and aggregates, while the purified proteins were in theflow-through. The LPS levels in the final purified preparations were<0.1 EU/mg (range 0.02-0.08), a very low level compatible withpreclinical studies³³. As shown in FIG. 11 , the purity of recombinantproteins was >95%, as assessed by silver-stained SDS/PAGE gel and wassimilar to that of the FDA-approved drug EYLEA.

The recombinant proteins were tested for their ability to inhibitmitogenesis stimulated by VEGF (10 ng/ml) in bovine choroidalendothelial cells. The recombinant proteins had inhibitory effects, withIC₅₀ values were in the range of −1 nM, except for V₁₋₂₋₄ and V₂₋₄,which were less potent (FIG. 12 ). Interestingly, EYLEA, even at thehighest concentration tested, inhibited no more than ˜80% ofVEGF-stimulated proliferation (FIG. 12 ). In contrast, the presentVEGFR-1 constructs, (except, V₁₋₂₋₄ and V₂₋₄), completely blockedVEGF-induced proliferation (FIG. 12 ). Binding assays documented theinteraction between the soluble VEGF receptors and the biotinylated VEGFand the ability of VEGF to displace binding (FIG. 13, 14 ).

To further define therapeutically relevant interactions, we sought toassess whether the recombinant proteins bind bovine vitreous in vitro.As illustrated in FIG. 14 , while EYLEA or control IgG had little or nobinding, the present proteins showed significant binding. The strongestbinders were V₁₋₂₋₃₋₃, V₂₋₃₋₃ and V₁₋₂₋₃₋₄ followed by V₁₋₂₋₃. V₂₋₃ hadintermediate binding characteristics, between EYLEA (or control IgG) andV₁₋₂₋₃₋₃. AVASTIN, a monoclonal antibody⁹ commonly used to treatintraocular neovascularization, also had little or no binding.

To determine whether vitreous-bound VEGFR-1 FC fusion may bebiologically active, plates were coated with bovine vitreous. Additionof V₁₋₂₋₃₋₃, but not EYLEA or control IgG, inhibited the ability ofexogenously added VEGF to stimulate endothelial cell proliferation (FIG.15 ).

The recombinant proteins were tested in the mouse CNV assay and comparedthem to control IgG or EYLEA. Each protein was injected intravitreallyat the dose of 2.5 μg one day before laser treatment. EYLEA was testedalso at 25 μg. V₁₋₂₋₃, V₂₋₃, V₁₋₂₋₃₋₃ and V₂₋₃₋₃ at the dose of 2.5 μgdemonstrated a degree of inhibition similar or greater to that achievedwith 25 μg EYLEA. However, none of the constructs containing D4demonstrated significant inhibition under the circumstances tested (FIG.16 ).

To determine whether heparin binding may translate in durabletherapeutic effects following a single administration, V₁₋₂₋₃₋₃, EYLEAor control IgG, were injected intravitreally (2.5 μg) 1 day, 7 days or14 days before the laser-induced injury. As shown in FIG. 17 , EYLEAresulted in a significant inhibition only when administered 1 day beforethe injury. However, V₁₋₂₋₃₋₃ resulted in a significant inhibition alsowhen administered 7 days or 14 days prior to the injury.

Disclosed are several novel VEGFR-1-Fc constructs evaluated in a varietyof in vitro and in vivo assays. To purify the recombinant proteins, amulti-step protocol was used. This was dictated to a large degree by therelatively low expression levels in transiently expressing 293 cells,requiring the addition of heparin to the media to improve release.However, the need to use heparin may be in part or entirely obviated bydifferent host cells (e.g., having a different composition of HSPG ormutants thereof) or by higher expression levels such as in amplified,stable cell lines.

All constructs, except V₂₋₄, potently neutralized the activity of VEGFand, at the same time, had strong heparin-binding characteristics, whichmay predict a long half-life following intravitreal administration. Theexample documents that these proteins bind to bovine vitreous. Thestrongest binders were V₁₋₂₋₃₋₃, V₂₋₃₋₃, V₁₋₂₋₃₋₄, followed by V₁₋₂₋₃.V₂₋₃ had significant but lower vitreous binding. Control IgG, EYLEA, orAVASTIN had instead minimal binding. One of the strongest vitreousbinders, V₁₋₂₋₃₋₃, was selected to test the hypothesis that a vitrealmatrix-bound VEGFR1 Fc constructs may be biologically active. As shownin FIG. 15 , in plates coated with bovine vitreous, addition ofV₁₋₂₋₃₋₃, but not EYLEA or control IgG, inhibited the ability ofexogenously added VEGF to stimulate endothelial cell proliferation.

Next the recombinant proteins were tested in the mouse CNV model fortheir ability to inhibit laser-induced neovascularization. EYLEA wasused as a positive control and human IgG1 as a negative control.Relatively low dose was chosen for in vivo testing, being best suited toreveal potency differences among the various constructs. Also, it hasbeen reported that intravitreal administration of relatively high dosesof antibodies of the IgG1 isotype may have off-target inhibitoryeffects, mediated by FcgRI (CD64) and c-Cbl, when injectedintravitreally 34. The dose employed should avoid such off-targeteffects and detect truly specific effects.

As shown in FIG. 16 , EYLEA resulted in an approximately 30% inhibitionat the dose of 2.5 μg and ˜50% inhibition at 25 μg. These findings arelargely consistent with the published literature. Saishin et al reportedthat the intravitreal injection of ˜5 μg aflibercept resulted in ˜30%inhibition of CNV area in the mouse³⁵. Indeed, the dose of 40 g iscommonly used to achieve a maximal effect in the mouse CNV model³⁶. Anunexpected finding of our study was the greater potency of some of ourconstructs: V₁₋₂₋₃, V₂₋₃, V₁₋₂₋₃₋₃ and V₂₋₃₋₃. Administering 2.5 μg ofthese constructs one day before the injury matched or even exceeded thelevel of inhibition achieved with 25 μg of EYLEA. The finding thatV₁₋₂₋₃₋₃, but not EYLEA, has significant effect on preventing CNV whenadministered 7 days or 14 days before the injury (FIG. 17 ), documentsthe durability of the effect and the therapeutic value.

An unexpected finding is that none of the constructs containing D4(V₁₋₂₋₃₋₄, V₂₋₃₋₄, V₁₋₂₋₄, V₂₋₄) resulted in marked inhibition in vivo(at least at the dose tested), in spite of the fact that these molecules(with the exception of V₂₋₄) demonstrated an ability to blockVEGF-stimulated mitogenesis in vitro. However, all of these constructsdemonstrated a propensity to form multimers or aggregates, as assessedby SDS/PAGE gel under non-reducing conditions (FIG. 11 ) or sizeexclusion chromatography (not shown). Although earlier work³⁷ identifiedD4 (together with D7) as a requirement for VEGFR-1 dimerization, sucheffect has been known to be ligand-dependent. Crystal structure studiesrevealed a loop in D4 responsible for such homotypic interactions²³. Itis conceivable that high concentrations and/or the forced dimerizationimposed by the Fc construct may result in ligand-independentinteractions, resulting in aggregation. In any event, aggregates are notdesirable pharmaceuticals given the possibility of inflammation andimmunogenicity^(38, 39) Therefore, an aspect of the present invention isthe identification of constructs having VEGFR-1 D2/D3, but not D4, inembodiments.

It is noteworthy that well-characterized Fc mutations, well known to theskilled in the art, that reduce effector functions could be usefuladditions to the invention in order to minimize antibody-dependentcytoxicity (ADCC) well as interactions with C1q and the initiation ofthe complement cascade⁴⁰.

In conclusion, aflibercept was designed to eliminate the heparin-bindingheparin domain in order to improve systemic half-life for oncologicalindications. The constructs described in the present study are insteaddesigned to promote binding and retention in the vitreous to ensure moresustained and therapeutically relevant interactions.

Methods

For construction of VEGFR-1_(ECD)-Fc expression plasmids, the nucleicacid fragments encoded the signal peptide and a variety of extracellularIg-like domains one to four²⁰ of VEGRF-1 (Gene ID: 2321) weresynthesized by GenScript USA Inc. The variety of the extracellularIg-like domain constructs is as follows: V123 contains D1, 2 and 3; V23,D1 and D2; V1234, D1, 2, 3 and 4; V1233, D1, 2, 3 and 3; V234, D 2, 3and 4; V124, D1, 2 and 4; V24, D2 and 4; F7 is ECD2, 2 and 3 and F8 isECD2 and 3. The synthesized fragments were inserted into pFUSE-hIgGl-Fcvector (InvivoGen, #pfuse-hgifc1) at EcoRI and BgIII sites, generatingthe plasmids containing the various Flt1 ECDs. Then, using PrineSTARMutagenesis Basal Kit (Takara, R046A), the interval amino acid R and S(BgIII site) between the ECDs and the Fc fragment were removed,generating the plasmids (F1-F8) expressing the fusion proteins of Flt1ECDs with a 227-amino acid human IgG1-Fc.

Transfection and Conditioned Media Preparation

The Expi293 expression system (Life technologies, A14524) was used togenerate the conditioned media for purification, according to themanufacturer's instructions. In brief, Expi293F™ Cells (ThermoFisher)were suspension-cultured in Expi293™ expression medium at 37° C. in ahumidified atmosphere with 8% CO2. When the cell density reached to 2.5million/ml, plasmids DNA and ExpiFectamine™ 293 reagent was mixed,incubated 5 min and added to the cells. The final concentration of theDNA and transfected reagent was 1 μg and 2.7 μl per milliliterrespectively. Five hours after transfection, 100 μg/ml Heparin (Sigma,H3149) and protease inhibitor cocktail, 1:400 (Sigma, P1860), were addedto the cells. 16 hours after transfection, enhancer reagents 1 and 2were added. Ninety-six hours after transfection, conditioned media wereharvested. Aliquots were tested for Fc fusion proteins concentrationsusing a human Fc ELISA Kit (Syd Labs, EK000095-HUFC-2) according to themanufacturer's instructions. Protease inhibitors were added (1:500) tothe bulk, which was stored at −80° C. until further use.

Purification of Recombinant Proteins

Pyrogen-free reagents were employed. Prior to use, columns and equipment(Akta Explorer System) were sanitized by exposure to 0.5 N NaOH forapproximately 45 minutes. Conditioned media from transfected cells wereadjusted to PBS, 0.01% polysorbate (PS) 20. PS20 was added to buffers atall steps. After centrifugation at 20,000×g for 30 minutes, supernatantswere subjected to protein A (PA) affinity chromatography using a Hi-TrapMabSeledt SuRe (5 ml, GE Healthcare). After loading, the column waswashed with 20 mM diethanolamine, pH 9.2, 1.2 M NaCl, prior to elutionwith 0.1 M citric acid, pH 3.0, which was immediately neutralized. ThePA elution pool was then diluted in 20 mM diethanolamine, pH 9.2, andapplied to Hi-Trap Q (5 ml, GE Healthcare) anion-exchange column. Thebound material was eluted with a gradient of NaCl. The flow-through,which contained the purified recombinant protein, was immediatelyadjusted to 20 mM Tris, pH 6.8, and then concentrated through binding toheparin-sepharose (Hi-Trap™-HS). After a wash with 0.2-0.45 M NaCl(depending on the construct), the recombinant VEGFR1 fusion protein waseluted with 1 M NaCl. The final polishing step consisted ofsize-exclusion chromatography (SEC), using, for example, Superdex 200Increase, 10/300 GL or Hi-Load 16/600 Superdex 200 μg, GE Healthcare.Finally, the proteins were buffer-exchanged by dialysis into 10 mM Tris,pH 6.8, 10 mM histidine, 1% threalose, 40 mM NaCl, 0.01% PS20. Todetermine endotoxin levels, ToxinSensor Chromogenic LAL Endotoxin AssayKit (GenScript, L00350) was used according to the manufacturer'sprotocol.

Cell Proliferation Assays

Bovine endothelial cell proliferation assays were performed essentiallyas previously described⁴¹. Log phase growing bovine choroidalendothelial cells (BCEC) (passage <10) were trypsinized, re-suspendedand seeded in 96-well plates (no coating) in low glucose DMEMsupplemented with 10% bovine calf serum, 2 mM glutamine, andantibiotics, at a density of 1000 cells per well in 200 μl volume.rhVEGF₁₆₅ (Peprotech) was added at the concentration of 10 ng/ml.Aflibercept (EYLEA) was purchased from a pharmacy. The inhibitors wereadded to cell at various concentrations, as indicated in the figures,before adding the ligands. After 5 or 6 days, cells were incubated withAlamar Blue for 4 hr. Fluorescence was measured at 530 nm excitationwavelength and 590 nm emission wavelength.

Solid-Phase VEGFR-1 Variant Binding Assays

Costar 96-well EIA/RIA stripwells (#2592, Corning Incorporated,Kennebunk, Me.) were coated overnight at 4° C. with purified VEGFreceptor proteins (250 ng/well) in coating buffer (Biolegend, San Diego,Calif., #421701). Nonspecific binding sites were blocked by incubatingthe strips with 2% BSA (Sigma, A6003) in PBS for 1 hour at roomtemperature (RT) after a single wash with ELISA wash buffer (R&D systems895003). Strips were then washed 3 times, followed by addingbiotinylated human VEGF₁₆₅ (G&P Biosciences, Santa Clara, Calif.) inassay diluent (Biolegend, #421203) alone or in combination with variousconcentrations of non-biotinylated human VEGF165 (R&D systems) at 37° C.for 2 hours. After three washes, bound biotinylated human VEGF₁₆₅ toVEGFR1 was detected by incubation with HRP Streptavidin (1:1000,Biolegend, #405210) for 1 hour at RT. Strips were washed 5 times beforeincubation with TMB high sensitivity substrate solution (Biolegend,#421501) for 30 min, and absorbance at 450 nm was measured after addingequal amount of stop solution (Biolegend, #77316). All experiments werecarried out in duplicate wells and repeated for at least two times.

In Vitro Binding to Bovine Vitreous

Bovine vitreous (InVision BioResource, Seattle, Wash.) was thawed at 4°C. Material was first diluted 1:1 with PBS, filtered through 0.22 mfilter, aliquoted and stored at −80° C. Total protein concentration ofbovine vitreous material was measured by Pierce BCA protein assay.Costar 96-well EIA/RIA stripwells were coated with bovine vitreous (1μg/well) for 4 hr at RT, followed by one wash with ELISA wash buffer.Nonspecific binding sites were blocked by adding 2% BSA in PBS for 1 hrat RT, followed by washing three times with 0.01% PBS-T. To each well,50 ul VEGFR1 or control proteins were added overnight at 4° C. Next day,plates were washed three times with 0.01% PBS-T, followed by incubatingwith 100 ul AP-conjugated goat anti-human Fc (1:2000, Invitrogen,#A18832)) for 1 hr at RT. Plates were further washed five times with0.01% PBS-T before adding 50 ul 1 step PNPP substrate (ThermoScientific, Rockford, Ill., #37621) for 15-30 min at RT. OD was measuredat 405 nm.

Effects of vitreous bound VEGFR1 on VEGF-stimulated endothelial cellproliferation in Costar 96-well EIA/RIA stripwells were firstUV-sterilized for 1 hr, followed by coating with 1 g/well bovinevitreous, diluted in PBS for 4 hr at RT. Plates were washed with PBSonce, blocked with 2% BSA at 4° C., and washed two times with PBS inbiosafety hood. Equal amounts of soluble receptors or control IgG wereadded to plates, diluted in PBS O/N at 4° C. (50 μl/well). Plates werethen washed once with PBS, followed by one wash with assay mediacontaining 10% BCS. 100 μl media was added to each well, followed byaddition of VEGF at 5 ng/ml or PBS only as no VEGF control. Plates wereincubated with VEGF or PBS for 1 hr, followed by adding 100 μl BCEC cellsuspension (final 2500 cells/well). 48 hrs later, proliferation wasmeasured by adding Alamar Blue.

Laser-Induced Choroidal Neovascularization (CNV)

Male C57BL/6J mice (6-8 week) were anesthetized with ketamine/Xylazinecocktail before laser treatment. CNV lesions were induced by laserphotocoagulation using a diode laser (IRIDEX, Oculight GL) and a slitlamp (Zeiss) with a spot size of 50 um, power of 180 mW and exposureduration of 100 ms.^(36, 42). Four laser burns were typically induced at3, 6, 9 and 12 o'clock position around the optic disc in each eye.Different constructs or IgG isotype control were injectedintravitreally, at the dose of 2.5 μg per eye, in a 1 μl volume. EYLEAwas used as a positive control at 2.5 or 25 μg. One day after injection,laser treatment was conducted and eyes were enucleated and fixed in 4%paraformaldehyde (PFA) for 15 min, 7 days after laser treatment. In aseparate set of studies, selected constructs were injected once 1 day, 7days or 14 days prior to laser treatment. Choroid-sclera complexes andretinas were separated and anti-CD31 immunofluorescence (IF) wasperformed to evidence the vasculature by whole mount staining of bothretina and choroidal tissues. For CD31 IF, rat anti-mouse antibody BD550274 was diluted 1:100 and incubated overnight at 4° C. After 4-hourincubation with a secondary anti-rat antibody (Life TechnologiesA11006), whole mounts were imaged at 488 nm. Quantification ofneovascularization in lesion area and vascular density in retina wascarried out by Image J. P values were assessed by Student's t test(significant change, p<0.05).

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What is claimed is:
 1. An anti-VEGF agent comprising a VEGF bindingportion operatively linked to a Fc-IgG, wherein the VEGF binding portioncomprises a VEGF binding domain consisting essentially of an IgG-likedomain 2 of VEGFR-1, a VEGF binding domain that is an IgG-like domain 3of VEGFR-1, and a VEGF binding domain that is an IgG-like domain 4 ofVEGFR-1.
 2. The anti-VEGF agent of claim 1, wherein the anti-VEGF agentcomprises the amino acid sequence of SEQ ID NO:
 11. 3. A pharmaceuticalcomposition comprising a therapeutically effective amount of theanti-VEGF agent of claim 1 and a pharmaceutically acceptable excipient.